Metabolic Engineering and Synthetic & Systems Biology Laboratory

Abhishek Murarka

Education

Project Description

Metabolic flux analysis (MFA) is one of the most important analytical tools in metabolic engineering. Through in vivo quantification of metabolic fluxes, it allows the systematic study of cellular responses to genetic and environmental perturbations. MFA based upon stoichiometry and extracellular measurements (conventional MFA, c-MFA) is a very useful, noninvasive, and cost-effective process that gives a first-pass analysis of the system that can be used as a basis for further studies. Carbon-13 labeling techniques, with analysis by NMR or GC/MS, have gained wide use in the metabolic engineering community for providing flux ratios in central carbon metabolism. This data, in conjunction with stoichiometry and extracellular measurements, provides a more rigorous interpretation of the network topology and flux values. We are currently using c-MFA and NMR-based MFA to study the metabolism of E. coli strains devoid of enzymes responsible for the dissimilation of pyruvate during anaerobic fermentation of sugars.

Pyruvate is a key precursor metabolite for the biosynthesis of building blocks in E. coli under both aerobic and anaerobic conditions. It gives, upon dissimilation, acetyl CoA, which is also an important biosynthetic precursor metabolite. Under anaerobic conditions and in the absence of external electron acceptors, pyruvate is also the most prominent intermediate metabolite for the synthesis of several fermentation products such as lactate, formate, ethanol, and acetate. The conversion of pyruvate to lactate is achieved via a fermentative pathway catalyzed by the enzyme lactate dehydrogenase. The conversion of pyruvate into acetyl-CoA is catalyzed by two different enzymes: pyruvate formate lyase (PFL, coded for by pflB gene) and pyruvate dehydrogenase (PDH, coded for by aceEF-lpdA genes). There is one more enzyme involved in the dissimilation of pyruvate; pyruvate oxidase (PoxB), a product of gene poxB. PoxB catalyses the decarboxylation of pyruvate to acetate and CO2 with the reduction of flavin adenine dinucleotide, FAD. The metabolism of pyruvate via PoxB is less efficient than the route via the PDH; however, the PoxB route is important for wild-type growth efficiency and responsible for a significant amount of pyruvate metabolism under aerobic conditions. However, its metabolic role (if any) under anaerobic conditions is still unknown. The objective of this work is to elucidate the physiological role of PFL, PDH, and PoxB in the metabolism of E. coli during anaerobic fermentation of sugars.